REAGENTS AND PROCESSES 541 



same time, and to aid in this the material to be fixed should be cut into the smallest 

 pieces compatible with the purpose of the study. 



Absolute alcohol or 95 per cent, alcohol penetrates the tissues quickly, and for 

 many subjects is an excellent fixative. Material fixed in alcohol does not, as a rule, 

 yield the best results with staining media, and such material should, as already 

 recommended, after sectioning and mounting on the slide, be placed in a I per cent, 

 solution of chromic acid for about twenty -four hours, and then washed for two hours 

 in water. Then the sections may be stained. 



Flemming's Mixture. The best all-around fixative is Flemming's mixture: 



1 per cent, chromic acid 16 parts, 



2 per cent, osmic acid 3 parts, 



Glacial acetic acid i part. 



A saturated solution of corrosive sublimate in strong alcohol penetrates quickly, 

 and tissues need to be left in it for only a few hours. The corrosive sublimate should 

 be washed out with alcohol, to which crystals of iodine have been added, until it 

 assumes a brown color. 



A concentrated solution of picric acid in alcohol is an excellent fixative for 

 aleurone grains in oily seeds. 



It is best to thoroughly wash out all fixatives before the process of hardening in 

 successively higher grades of alcohol is begun. This is accomplished for those fixa- 

 tives which do not contain corrosive sublimate by washing in running water for 

 about six hours, or over night. Fixatives containing corrosive sublimate should be 

 washed in alcohol containing iodine, as above suggested. 



The material to be fixed should be made to sink at once in the fixative. In alco- 

 holic fixatives this will occur very quickly without any special means to accom- 

 plish it, but for aqueous fixatives it may be necessary to first pump the air from 

 the tissues. This always insures a better injection of the tissues by the fixative, 

 and is to be recommended for most material. Any sort of air-pump will answer. 

 A bicycle-pump with the valve reversed does excellent service, and has the ad- 

 vantage of portability, so that it may be carried into the field where material may 

 be fixed as gathered. In pumping out the air the material should be immersed in 

 0.5 per cent, chromic acid if Flemming's fixative is to be used, and after the material 

 has been made to sink in this, the chromic acid should be immediately replaced 

 by the intended fixative. In most aqueous fixatives, such as Flemming's, the ma- 

 terial should be left for about forty-eight hours. In the alcoholic fixatives the 

 material may be left from a few hours to twenty -four hours. With some material 

 the best results are obtained by using the fixatives hot. This is true of many 

 sporangias and zygospores, which are very difficult of penetration by the fixative. 

 Since the osmic acid is quite volatile, special precautions must be taken in heating 

 fixatives of which it forms a part. Good results have been obtained in the follow- 

 ing manner: The fixative (preferably Flemming's) was poured into a tall test-tube 

 to the height of a few centimeters. Into this was placed the material to be fixed, 

 and the tube was tightly stoppered. Then the test-tube was immersed in a vessel 

 of boiling water to the depth of the fixative, where it remained for about three 

 minutes, or until the fixative began to show signs of boiling; then the test-tube 

 was removed and plunged into cold water. After cooling, the stopper was re- 

 moved and the material then sank in the fixative. Since the long test-tube ex- 

 tends for some distance above the boiling water, the upper part of the tube remains 

 sufficiently cool to condense the vapors of the fixative as they are formed, and in 

 this way any material change in the composition of the fixative is prevented. The 

 material should remain in the cool fixative for about twenty-four hours, and then 

 should be washed out and dehydrated in the usual manner. 



